High-resolution NMR spectroscopy of molecules encapsulated in low-viscosity fluids

ABSTRACT

The present invention provides a method for reducing the correlation time for global tumbling for analysis of proteins and other macromolecules by NMR spectroscopy. The method comprises placing a macromolecule, hydrated and without significant structure perturbation, within a reverse micelle in a suitable fluid medium of low viscosity. A high pressure NMR cell for use with the method of the present invention is also disclosed.

This application is a divisional of U.S. patent application Ser. No.09/306,906 filed on May 7, 1999, now U.S. Pat. No. 6,198,281, which is acontinuation-in-part of U.S. patent application Ser. No. 08/967,996,filed on Nov. 12, 1997, now U.S. Pat. No. 5,977,772, and which claimsthe priority of a provisional application serial No. 60/084,702 filed onMay 8, 1998, the disclosures of which are incorporated herein byreference.

This work was supported by grant number GM35940 from the NIH. Thegovernment has certain rights in-the invention.

FIELD OF THE INVENTION

This invention generally relates to the field of NMR spectroscopy. Morespecifically, this invention relates to a process and apparatus for ahigh-resolution NMR spectroscopic analysis of molecules, particularly,macromolecules.

BACKGROUND OF THE INVENTION

Nuclear magnetic resonance (NMR) spectroscopy continues to play acentral role in the characterization of the structure and dynamics ofproteins, nucleic acids, carbohydrates and their complexes. Over thepast fifteen years there have been staggering developments in NMRtechniques and supporting technologies such that the comprehensivestructural characterization of 20 kDa proteins is becoming almostroutine. Second only to crystallography, NMR spectroscopy provides anunparalleled view of structure and it remains second to none in itsability to examine dynamic phenomena. NMR also provides a unique avenueto monitor the full structural and dynamic effects of changes intemperature, solution conditions and the binding of small and largeligands.

Application of NMR to analysis of Protein Structure

The size of proteins that can be analyzed by modern NMR techniques hasdramatically increased over the past decade. Coupled with theintroduction of heteronuclear (Sorensen et al., 1987, Progr. NMRSpectroscopy 16:163-192.) and ultimately triple resonance (Kay et al.,1990, J. Magn. Reson. 89:495-514.) spectroscopy, was the wide spread useof recombinant technologies to introduce NMR-active isotopes intoproteins and nucleic acids (LeMaster, 1994 Progr. NMR Spectroscopy26:371-419; McIntosh & Dahlquist, 1990, Q. Rev. Biophys. 23:1-38). Withthe development of multinuclear and multidimensional capabilities, NMRis now able to confidently, efficiently and comprehensively deal withsmall proteins with significant spectral complexity. However, for largeproteins, increasing size brings with it several important limitationswhich unfortunately compound each other. This severely limits the sizeof a protein that can be efficiently approached by modern NMRtechniques.

One limitation is that increasing size leads to slower tumbling andcorrespondingly shorter spin-spin relaxation times. The basic engine ofNMR spectroscopy of proteins, the triple resonance technology, begins tofail. As lines broaden, basic sensitivity also becomes a limiting issue.Another limitation is that increasing size leads to increasingly complexspectra: Spectral degeneracy complicates the assignment process andrenders assignment of NOEs to parent hydrogens problematic.

One way to reduce the problems posed by the size and hence complexity ofthe protein is to reduce the limitations presented by short spin-spinrelaxation times. As already mentioned, increasing size leads to shorterspin-spin relaxation times. Since the coherence transfer processesunderlying current triple resonance-based assignment strategies aretime-dependent, these approaches begin to fail with proteins ^(˜)30 kDaand larger. Random partial or perdeuteration has been used tosuccessfully reduce the dipolar field such that high resolution ¹⁵N-HSQCspectra can be obtained (LeMaster, 1994, supra). Unfortunately,perdeuteration drastically limits the structural information availablefrom the NOE. Fractional deuteration also has its own problems withrespect to sensitivity and its limited applicability as a generalsolution to the dipolar broadening displayed by proteins above 35 kDa.Spectroscopic solutions are also appearing. Some find their roots in thesteady improvement in the use of the rotating frame to provide for moreefficient isotropic mixing for coherence transfer. One very recentadvance is the selection of the narrow multiplet component arising dueto the (fortuitous) cancellation of dipole-dipole coupling and chemicalshift anisotropy in ¹⁵N-¹H correlation experiments (Pervushin et al.,1997, Proc. Natl. Acad. Sci., USA, 94:12366-12371). This particularapproach will not relieve the limitations in other contexts. In short,though these and other current approaches are extremely helpful, they donot appear to be generally applicable nor generally robust.

Reverse Micelle Technology

Reverse micelles form spontaneously as transparent solutions in a lowpolarity liquid and are thermodynamically stable assemblies ofsurfactant molecules organized around a water core. Reverse micelleswere the subject of extensive attention in the 1980s as potentialdevices for a range of applications including separations,chromatography and reaction processes (Goklen & Hatton, 1985,Biotechnology Progress, 1:69-74). More recently, they have become thefocus of further attention in the context of hosting various chemicalreactions in solvents with low environmental impact such assupercritical carbon dioxide (Johnston et al., 1996, Science271:624-626).

The size and stability of reverse micelles is dependent upon the amountof water loading. Water loadings have been described that yield stablereverse micelles of AOT in a variety of long and short chain alkaneslarge enough to accommodate proteins (e.g., Frank & Zografi, 1969, J.Colloid Interface Sci. 29:27-35; Gale et al., 1987, J. Am. Chem. Soc.109:920-921; Fulton & Smith, 1988, J. Phys. Chem. 92:2903-2907; Fultonet al., 1989, J. Phys. Chem, 93:4128-4204).

For the analysis of protein structure, while solid state NMR methodscontinue to show great progress and recent successes like thedetermination of the gramicidin channel illustrate the potential ofthese approaches (Ketchem et al., 1996, J. of Bimolecular NMR 8:1-14),solution NMR methods are easier to employ. However, the difficulty ofdealing comprehensively with large proteins in a general manner remainsas a significant limitation to applying solution NMR methods to therapidly growing list of proteins being discovered by the molecularbiology community.

Thus, there is an ongoing need for novel techniques and approaches forextending the technique of solution NMR to proteins, especially, largerproteins, and other macromolecules. For example, fully 25% of known openreading frame sequences appear to code for membrane proteins and over50% code for proteins that are beyond the size accessible by currentsolution NMR methods.

SUMMARY OF THE INVENTION

The spin-spin relaxation time (T₂) is often the dominant limitation withrespect to the successful application of modern multinuclear andmultidimensional NMR spectroscopy to a particular protein. In theabsence of unrestricted internal motions, T₂ increases as thecorrelation time for global tumbling tau(m) decreases. Increased T₂results in a higher signal to noise ratio and the effectiveness of thecurrently available NMR techniques decreases.

The present invention provides a method for reducing the correlationtime for global tumbling tau(m) for analysis of molecular structure byNMR. The method comprises placing a macromolecule, hydrated and withoutsignificant structure perturbation, within a reverse micelle rapidlytumbling in a suitable fluid medium. This is accomplished byencapsulating the protein of interest within the water cavity of areverse micelle. The reverse micelle is in turn solvated by a lowviscosity alkane solvent. Thus, although the micelle is larger than theencapsulated hydrated molecule, it tumbles faster in the alkane solventthan the molecule will in water. The use of a very low viscosity alkanesolvent rests on the ability to prepare and maintain the sample undermodest pressure (ranging up to 50 atm, depending on the solvent). Underthese conditions, some alkane solvents have sufficiently low viscositiesto provide for short correlation times for molecular tumbling of theentire reverse micelle. Also disclosed in the present invention is aunique pressure cell for pressurizing the sample using the high pressurecell technique for solution NMR spectroscopy.

Accordingly, an object of the present invention is to provide a methodfor high resolution NMR spectroscopy by reducing the effective tumblingtime of molecules. This method is particularly useful formacromolecules.

Another object of the present invention is to provide a method for highresolution NMR spectroscopy of proteins by reducing the effectivetumbling time.

Another object of the present invention is to provide a method for highresolution NMR spectroscopy of macromolecules at low temperature.

A further object of the present invention is to provide an apparatus forthe analysis of molecules by NMR spectroscopy by using the method of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a reverse micelle containing a watersoluble protein.

FIG. 2 is an illustration of the phase transfer method forsolubilization of water core of reverse micelles formed in organicsolvent.

FIG. 3 is a plot illustrating the maximum solubility of reducedcytochrome c in AOT reverse micelles in isooctane(2,2,4,-trimethylpentane) or n-pentane.

FIG. 4 is a plot of comparison of diffusion rates of ubiquitin andlysozyme in water to the diffusion rates of “empty” AOT micelles inpentane and cytochrome c loaded micelles in pentane.

FIG. 5 is a schematic illustration of solubilization of an integralmembrane protein.

FIG. 6 is a schematic illustration of a cross section of the pressurecell used in the method of the present invention.

FIG. 7 is a schematic illustration of a cross section of the housing ofthe pressure cell.

FIG. 8 is a schematic illustration of a cross section of the cover ofthe pressure cell.

FIG. 9 is a schematic illustration of a cross section of the sample tuneof the pressure cell.

FIG. 10 is a ¹⁵N-HSQC spectrum of recombinant human ubiquitin inAOT/water/pentane reverse micelles in an 8 mm probe in a standard tube.

FIGS. 11a and 11 b is a representation of the correlation of amide ¹H(FIG. 11a) and ¹⁵N (FIG. 11b) chemical shifts of recombinant humanubiquitin in aqueous buffer with those of the protein encapsulated in areverse micelle dissolved in pentane.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel method and apparatus for NMRspectroscopy of molecules. This method is particularly suitable forproteins and other macromolecules. The method utilizes reducing theeffective tumbling time of molecules during NMR measurements byproviding an environment of low viscosity for them. While examples areprovided herein with particular reference to proteins, it will beapparent to those skilled in the art that the method of the presentinvention is suitable for other macromolecules also including, but notlimited to, nucleic acids and carbohydrates.

A high pressure cell for conducting NMR measurements is also provided.The spin-spin relaxation time (T₂) is often the dominant limitation withrespect to the successful application of modern multinuclear andmultidimensional NMR spectroscopy to a particular protein. The spin-spinrelaxation time decreases as the tumbling time of proteins during NMRmeasurement increases. The tumbling time of proteins is in turn,linearly related to the viscosity of the solvent.

In the present invention, solvents of low viscosity have been used toprovide an environment of low viscosity for the protein. A “lowviscosity fluid” or a “low-viscosity solvent” for the purposes of thespecification and claims means any hydrophobic solvent with viscositylower than about 300 μPa.s. An example of low viscosity solvent is ashort chain alkane whose intermolecular interactions are limited toLondon forces. Short chain alkanes suitable for the method of thepresent invention include, but are not limited to, straight or branchedalkanes containing up to eight carbons. Thus, short chain alkanesinclude ethane, propane, butane, pentane, hexane, septane, octane andtheir corresponding branched alkanes. Ethane, propane and-butane aregases at room temperature and pressure but are liquefied by modestpressures (Table 1). The viscosity of ethane, propane, butane andpentane is shown in Table 1. Other examples of low viscosity solventsinclude carbon dioxide, and halocarbons such as CF₄ and CHF₃ that aregases at room temperature and liquids at modest pressures used in NMRtechniques.

TABLE 1 Viscosity of Short Chain Alkane Fluids at 300 K Ethane PropaneButane Pentane Water Viscosity 35 97 158 ˜220 850 (μPa · s) Pressure 4.71.05 0.40 0.10 0.10 (MPa) Note: 1 MPa = 10 bar = 145 psi ˜10 atm; 1 μPa· s = centipoise; The figure for pentane results from an interpolation.

While many liquids have low viscosity, the use of such liquids in NMRhas heretofore not been possible since protein molecules could not beplaced directly in these solvents under the pressures required for NMR.

To place the protein molecule in an environment of low viscositysolvents without affecting their structural integrity, the reversemicelle technology was used. To place a protein in a hydrophobic solventsuch as alkane solvents, the protein molecules are encapsulated withinthe water cavity formed by so-called reverse micelles. The reversemicelles are formed by suitable'surfactants. For forming reversemicelles, it is preferable to use surfactants that have branched orbulky tails and small head groups so that the head groups are directedtowards the cavity of the micelle. Suitable surfactants include, but arenot limited to, dioleylphosphoric acid (DOLPA) and sodiumbis(2-ethylhexyl)sulfosuccinate (AOT). The size and stability of reversemicelles is dependent upon the amount of water loading. The commonlyused surfactant, AOT has been extensively studied in this respect for avariety of organic solvents and is commonly used in the art to constructreverse micelle systems having a significant internal aqueous core. AOTreverse micelles have average aggregation numbers ranging between 45 and70, depending on the amount of water loading in the interior. FIG. 1 isa schematic illustration of an AOT reverse micelle containing a watersoluble protein. Such systems have been developed for a large number ofproteins and are well known to those skilled in the art. Thus, manyproteins exceeding 50 kDa in size have been successfully solublized byreverse micelles in organic solvents (Luisi et al., 1988, BiochemBiophys Acta 947:209-246; De et al., 1995, Adv. Colloid Interface Sci.59:95-193; Goto et al., 1997, Biotech. Bioeng. 54:26-32; Johnston etal., 1996, Science 271:624-626).

While protein containing reverse micelles can be prepared by any methodknown to those skilled in the art, it is preferable to use a two-steppassive phase transfer process whereby empty reverse micelles areprepared and then subsequently loaded with protein. The process by whichhydrated proteins are encapsulated within reverse micelles in thelow-viscosity solvent involves liquefaction of the solvent, solvation ofthe surfactant (AOT) in the solvent; and transfer or distribution of thehydrated protein into the AOT-solvent phase via encapsulation.

While variations of the basic approach will be apparent to those skilledin the art, two variations, by way of illustration, are presentedherein. In both cases, the solvent is first liquefied within a pressurecell in the presence of AOT. Concurrent stirring (for example, by usinga small conventional magnetic stir-bar) facilitates preparation of ahomogenous solution of reverse micelles in the liquefied solvent. Thesolution of AOT reverse micelles is transferred into a second mixingcell which has been preloaded with the hydrated protein. The solution ofAOT reverse micelles and the hydrated protein are then mixed until thedesired loading of protein is achieved. The protein-solvent-AOT systemis then transferred into the NMR cell. Alternatively, the solvent-AOTphase is delivered directly into the NMR cell, which has been preloadedwith the hydrated protein. The hydrated protein is combined with thesolvent-AOT phase using gentle agitation that may be aided by a freestanding glass capillary filled with a lock solvent (D₂O).

Liquefaction of the solvent at a given temperature is achieved byraising the pressure of the system above the liquefaction pressurespecified at the gas-liquid phase boundary by the pressure-temperaturephase diagram for the solvent. Elevated pressure may be generatedthrough standard means, for example, by using a pressure generator orthe use of an inert gas. Transfer of solution of reverse micelles to asecond mixing cell or to the NMR cell is readily accomplished bycreating a small pressure difference (for example about 10 p.s.i.)between the vessels.

Solubilization of proteins in reverse micelles can be followed byoptical methods. Fluorescent mutants can be used for proteins that havelow absorbance and fluorescence. For example, a highly fluorescentmutant P45W has been reported for ubiquitin (Khorasanezadeh et al.,1993). A key component of the present invention is the degree to whichproteins can be solubilized in a reverse micelle-containing organicphase. Though most examples of protein solubilization in reversemicelles have been undertaken at sub-100 μM concentrations of protein,there are many examples where sub-mM concentrations have been obtained.Indeed, it appears that low concentrations of solubilized proteins havebeen used by choice (for example, to avoid self-quenching offluorophors) and does not reflect an inherent limitation. For example,the transfer of cytochrome c from neutral aqueous solutions of modestionic strength to 250 mM AOT in isooctane has been shown to occur withinminutes of mixing (Goklen & Hatton, 1985, supra). Final concentrationsof cytochrome c approaching 0.25 mM in the organic micellar solutionwere easily obtained. Essentially complete transfer could be obtained.Thus, by varying the relative volumes of the aqueous and organic phases,the protein could be concentrated (V_(aq)/V_(orq)>1) or diluted(V_(aq)/V_(org)<1) for use in the method of the present invention.Transfer efficiencies and indeed the direction of transfer are known tobe dependent upon the ionic strength of the aqueous solution (Goklen &Hatton, 1985, supra). Thus, as is known to those skilled in the art,solubilization of proteins in reverse micelles can be selectivelycontrolled by the choice of surfactant structure (e.g., cationic versusanionic), manipulation of pH and ionic strength and variation ofsurfactant concentration so that spectroscopic properties of thesolvated proteins are essentially those of the water-solubilizedprotein.

The radius of a reverse micelle is determined by the width of thesurfactant shell and the volume of the water pool and protein enclosedtherein. For example, the radius of an empty AOT reverse micelle isabout 15A. The absolute surfactant concentration, and the relative watercontent should be chosen carefully as described by Gardner et al.,(1997, Biochemistry, 36:1389-1401), which disclosure is hereinincorporated by reference, so that the protein filled reverse micelleradius corresponds to the simple sum of the hydrated protein's effectiveradius and the chain length of the surfactant. In one embodiment,encapsulation of a spherical 50 kDa protein with a spherical hydratedradius of 26 Å will result in a minimal reverse micelle of about 41 Å.The corresponding molecular tumbling correlation time of the reversemicelle would be about 60 ns in water, about 11 ns in butane, about 7 nsin propane and about 2.5 ns in ethane. The resulting increase in T₂according the method of the present invention makes proteins amenable tohigh resolution NMR. In another embodiment, encapsulation of a 100 kDaprotein (spherical hydrated radius of 33A) would have a correspondingreverse micelle radius of about 48 Å and would have a molecular tumblingcorrelation time of about 95 ns in water, about 18 ns in butane, about11 ns in propane, and about 4 ns in ethane. These correlation timevalues are a significant improvement over the corresponding values inwater directly which are 15 ns and 31 ns for the 50 kDa and the 100 kDaproteins respectively.

The use of low viscosity solvents like butane, propane or ethane as aliquid or supercritical fluid at or near room temperature requires theability to pressurize the sample. In the context of NMR spectroscopy,this presents several challenges. A, simple high pressure device thatwill allow state-of-the-art NMR spectroscopy to be safely carried out onproteins at intermediate pressures, up to 3,000 psi has been developed.These pressures will allow the prescribed reverse micelle approach to beused without great cost or difficulty and with the ability to employ themost powerful aspects of modem NMR spectroscopy of proteins. The designphilosophies underlying the device employed are distinctly differentthan those required for extremely high pressure and allow the use ofstandard bore magnets and the state-of-the-art commercial instrumentwithout significant risk or need for modification.

It must be emphasized that the pressures required for the method of thepresent invention are far below those required to detectably perturb thestructure of a native-state protein as shown in Table 3.

TABLE 3 Typical Pressures for macromolecular transitions^(a) and alkaneliquefaction^(b) Pressure (kbar) Enzyme inactivation 0.10-0.50 Liganddissociation 0.10-1.0  Protein-protein dissociation 1.0-2.0 DNA helixdissociation 2.0-3.0 Protein-DNA dissociation 2.0-3.0 Proteindenaturation (3° structure) 4-5 Protein denaturation (2° structure) 10-100 Short chain alkane liquefaction Ethane <0.050 Propane <0.011Butane <0.004 ^(a)Robinson & Sligar (1995, Methods Enzym. 259:395-427)^(b)Younglove and Ely (1967, J. Phys. Chem. Ref. Data 16:577-769)

An important aspect of the present invention is that because the samplesof the present method do not contain high salt and water content oftypical biological samples, they can be used in NMR probes having acooled or cryogenic probe head (U.S. Pat. No. 5,889,456). Such probeshave the advantage of low noise and high sensitivity. While the analysisof proteins, prepared by conventional methods, that contain high saltand water (commonly termed as “lossy” ) at low temperatures isproblematic, the present invention provides protein samples that areamenable to low temperature analysis.

Various other objects, features, and advantages of the present inventionwill become apparent from the following detailed description, taken inconjunction with the accompanying drawings.

EXAMPLE 1

This embodiment illustrates the preparation of protein-reverse micellesamples. Preparation of reverse micelles are known to those skilled inthe art. Lyophilized protein is dissolved in an appropriate volume ofaqueous buffer to achieve the desired water loading, w_(o) (mole ratioH₂O/surfactant), for the concentration of surfactant used. The waterloading parameter, w₀, represents a compromise between minimizing theaverage protein/micelle radius in pentane solution and having enoughwater (buffer) to solubilize the protein. Too much water in the micelle,i.e. typically, a w_(o) of greater than 30 will lead to a visibleprecipitate, while too little water i.e., typically a w_(o) of less thanabout 5, will lead to insufficient hydration and protein denaturation.As is known to those skilled in the art, optical water loading can bedetermined empirically.

The solubilization of proteins in reverse micelles dissolved in alkanesis dependent upon ionic strength, pH, and AOT concentration. Forexample, ethane, propane. and butane are known to those skilled in theart to support formation of homogeneous and stable solutions of highconcentrations of AOT reverse micelles. Solvent density appears to be akey factor in the capacity of a given alkane to support highconcentrations of reverse micelles. A rough estimate of the behavior ofthe reverse micelle system for each alkane with each protein can bedetermined by surveying their behavior in the classical AOT-isooctanesystem using UV/V absorption and fluorescence spectroscopy.

One method of solubilization of water-soluble proteins in the water coreof reverse micelles formed in organic solvent is the phase transfermethod and is illustrated in FIG. 2. The simple phase transfer methodworks well with isooctane where the protein is transferred from anaqueous layer to the organic layer. This method can be used for butanewhere only very modest pressures are required to maintain a liquidstate. Propane and ethane can be liquefied over a known amount of water,protein and surfactant. This allows for precise control of the watercontent. Solubilization of these three alkanes can be carried out bymethods known to those skilled in the art. For example, solubilizationcan be achieved by employing a so-called supercritical fluid (SCF) pump(Kloehn) to liquefy and transfer. A three window optical cell capable ofboth absorption and fluorescence spectroscopy at pressures up toapproximately 50 bar can be used.

In one illustration of this embodiment, ubiquitin reverse micelles wereprepared in AOT as the surfactant and pentane as the solvent. A sampleprepared with 2.0 ml of 75 mM AOT in pentane required 26.4 μL of aqueousbuffer to place w₀ at 10. To prepare a reverse micellar solution ofubiquitin, a 50 mM sodium acetate buffer at pH=4.5 containing 250 mMsodium chloride was used. The pH is chosen such that a net positivecharge is kept on the protein (pI˜7) which facilitates transfer of thehydrated protein into the AOT reversed micelles which have negativelycharged head groups on their interior. Sodium chloride is added tominimize strong interactions between ubiquitin and the head groups. Thesolubilized/wetted protein is transferred to a screw top NMR tube with ateflon septum, and premixed pentane-AOT solution is added to it with afew gentle shakes. A cloudy solution results which clears within 30minutes. A lock solvent such as benzeine-d6 is added (generally 7-10% ofthe total volume). Alternatively, a coaxial capillary containing D2O asa lock solvent can also be used. Finally, a vortex plug is placed at theair/solvent interface and the tube is properly sealed.

In another illustration of this embodiment, by using the method ofsolubilization disclosed herein, cytochrome c was solubilized in AOTreverse micelles in isooctane or pentane. The maximum solubility ofreduced cytochrome c in AOT reverse micelles in isooctane(2,2,4-trimethylpentane) or n-pentane is shown in FIG. 3. Samples wereprepared at room temperature by phase transfer from 0.1 M KCl, 50 mmpotassium phosphate solutions of cytochrome c into the indicated AOTsolution and assayed optically.

Solubilization of other proteins in reverse micelles can be carried outsimilarly. Preferably, there should be one protein molecule in a reversemicelle. However, the ability to control the number of protein moleculesthat are encapsulated per reverse micelle allows the use of the methodof the present invention to analyze protein aggregates such as prionproteins or amyloid proteins.

EXAMPLE 2

The hydrodynamic performance of the reverse micelle system was confirmedusing gradient diffusion methods of Altieri et al., 1995 (J., Am. Chem.Soc. 117:7566-7567), which method is hereby incorporated by reference.The apparent diffusion rates of ubiquitin in water (Mr 8.5 kDa) wascompared to the diffusion rates of “empty” AOT micelles in pentane andcytochrome c loaded AOT micelles in pentane. FIG. 4 shows the pulsedfield gradient NMR self-diffusion measurements of the translationaldiffusion of ubiquitin in water, lysozyme in water, AOT reverse micellesin pentane, and cytochrome C-AOT reverse micelles in pentane at 20 C.Tie fitted translation diffusion constants are 9.6×10⁻⁶, 7.3×10⁻⁶,4.0×10⁻⁶, and 1.9×10⁻6 cm²/sec, respectively.

As seen in FIG. 4, at 20° C., ubiquitin in water and the “empty” AOTreverse micelles in pentane have diffusion,rates differing by a factorof about 2. The “empty” AOT reverse micelles and cytochrome c loaded AOTmicelles in pentane also differ in diffusion rates about a factor of 2.The translation diffusion (Dtrans) constant for a sphere is given by theEinstein-Sutherland equation as:

D _(trans) =kT/f=kT/6Πηr _(h)

At this temperature, water and pentane differ in viscosity by about 4fold. This suggests that the effective radius of the “empty” AOT micellein pentane is about 1.6 times that of hydrated ubiquitin. A surprisingresult is the observation that the “empty” AOT reverse micelle has adiffusion constant approximately twice that of an AOT reverse micelleloaded with cytochrome c which is completely consistent with onecytochrome c molecule per micelle. These data demonstrate the ability ofusing the method of the present invention to give cytochrome c (in areverse AOT micelle in pentane) a faster translational (and presumablyrotational) diffusion rate than ubiquitin (in water), a smaller protein.

EXAMPLE 3

This embodiment illustrates the use of the method of the presentinvention for NMR analysis of proteins having exposed hydrophilic andhydrophobic components like integral membrane proteins. The reversemicelle carries with it other important potential applications. Incontrast to micelles of surfactants in water, the water containingreverse micelle in an organic solvent can offer two solvents to thesolubilized protein. This provides a potential route to thesolubilization of integral membrane proteins where the aqueous phasewould solubilize the hydrophilic component while the alkane phase to beused here would solubilize the integral membrane component. Thesurfactant would act as a bridge between the two phases, one organic andone aqueous, bridged by the reverse micelle surfactant to solubilize anintegral membrane protein. This is schematically illustrated in FIG. 5.The case on the left corresponds to a protein which has only onehydrophilic face while the case on the right corresponds to a proteinwhich normally completely spans the lipid membrane bilayer.

In addition, the present method can also be used to improve theresolution of proteins less than 25 kDa in size. For example, thesignificantly decreased linewidths will allow resolution of weakcouplings in E. COSY experiments directed at quantification of torsionangles. Similarly, lengthened T₂ times will allow more comprehensivelong range correlation analysis.

EXAMPLE 4

This embodiment illustrates the construction of a high pressure cell foruse with the method of the present invention. The pressure cell for theinvention should meet several criteria. First, as only sub-mMconcentrations of protein and other macromolecules are likely to beroutinely solubilized by solutions of reverse micelles, it is preferableto use the larger active volume of an 8 mm triple resonance probe tocompensate. Second, the NMR cell must be RF transparent, of NMR quality(good lineshape), and reliable at pressures of 50-100 bar. Third, theNMR cell must be accommodated by a standard triple resonance NMR probeand have the capability of “tuning” the pressure of the sample, i.e., becontinuously variable.

The relatively low pressures required for the application described hereopens up the number of materials that could be used. For high pressureNMR cells which must perform at pressures above 1.2 kbar, single crystalsapphire tubes need to be used. However, consideration of the geometryof the 8 mm tube makes use of single crystal sapphire problematic. Thereare several materials which have equivalent or superior tensilestrengths that can be precisely machined to such a geometry. Zirconiumoxide (i.e., hot isostatically pressed Zirconia) and silicon nitride aretwo examples. In a preferred embodiment, zirconium oxide is used. An 8mm o.d./6 mm i.d. tube would have a burst pressure well above 300 barand an active volume ^(˜)50% over that of a conventional 5 mm tube. Thetube is sealed to the housing by the self-sealing action of an embeddedO-ring and a washer. Similarly, the cover is sealed to the housing bythe self-sealing action of an O-ring. Thus, the use of glue or otheradhesives is avoided.

A schematic illustration of one design of the “high” pressure NMR cell10 for an 8 mm triple resonance probe is shown in FIG. 6. The BeCuhousing 12 is mated with a threaded top 14. The top has two portssuitable for high pressure fittings. The top port acts as an inlet forliquid alkane and the bottom port acts as an inlet for sample or as anoutlet for discharging gas while filling with liquid. The tube 16 isself-seals under pressure against the collar of the housing by a boronnitride washer 36 and an embedded O-ring 40. The housing self-sealsunder pressure against the threaded top by an O-ring 26 placed in acollar between the housing and the threaded top. The tube dimensionsshould be such that the tube will fit into the particular probeselected.

As shown in FIG. 7, housing 12 is a stepped cylindrical tube. It is madeof a non-magnetic metal with a high tensile strength. Suitable materialsfor the housing include, but are not limited to, stainless steel,titanium alloys, and beryllium copper alloys. In a preferred embodiment,the housing is made of Be—Cu alloy. Housing 12 has a wide upper section18 and a narrow lower section 20. Upper section 18 has an internallytapped well 22 to receive cover 14. The bottom corners of the well havea two-step edge. On the lower step 24 is housed O-ring 26 which providesa tight self-seal between cover 14 and upper section 18. Housing 12 hasthree bore sections, first bore section 28, second bore section 30, andthird bore section 32. Second bore section 30 is narrower than firstbore section 28. Third bore section 32 is wider than either first boresection 28 or second bore section 30. The sudden transition from secondbore section 30 to third bore section 32 provides a flat surface orcollar 34 which forms a sealing surface for sample tube 16. Washer 36 isplaced on collar 34 to provide a pressure seal between housing 12 andsample tube 16 on the load bearing side. In a preferred embodiment, thewasher is made of boron nitride. This washer also provides a cushion toallow for minor imperfections in the material of the sample tube or theBe—Cu collar 34. The diameter of third bore 32 is such that sample tube16 fits snugly in it. Lower section 20 also has groove 38 to captureO-ring 40 which forms a pressure seal with sample tube 16.

Cover 14 is made of the same material as the housing and has twointernal bores 56 and 58 (FIG. 8). When screwed into housing 12, cover14 fits into internally-tapped well 22. Cover 14 has top section 42,middle section 44, and lower section 46. The outer surface of middlesection 44 is threaded so as to be screwed into internally-tapped well22 of housing 12. The transition of the outer surface of middle section44 to lower section 46 is angled so as to capture O-ring 40 against thecorner of lower step 24 when cover 14 is screwed into housing 12. Anyangle such that the O-ring is not pinched or cut when installing thecover 14 into. housing 12 can be utilized to house the O-ring againstthe wall of lower step 24. In a preferred embodiment, this angle isbetween about 10° to about 70°. At the top of cover 14 the bores 56 and58 have threaded openings 57 and 59, respectively configured to receivestandard fittings. Cover 14 also has a step at the end of bore 58. Thisstep creates an opening 61 between the top of tube 16 and the step 60when installed. This opening acts as a collector for gas that separatesfrom the liquid phase.

The internal bore of tube 16 is of the same diameter as the internalbore of cover 14. Sample tube 16 is closed at one end, has main section50, and flange 52 at the open end. The outer diameter of main section 50is such that it will fit snugly in second bore section 30. When thesample tube is placed in the housing, flange 52 rests on collar 34.

In one embodiment, a pressure cell was constructed based on the interiordimensions of a standard Nalorac or Varian 8 mm probe and a standardOxford high. resolution shim stack assembly. Averting to FIGS. 6-9, thehousing has a first bore section 28 of 0.355″ diameter which transitionsinto a 0.3251″ second bore section 30 and then into a 0.503″ third boresection 32. The transition from the 0.325″ bore to the 0.503″ sectionprovides a flat surface or collar 34 on which a boron nitride washer isplaced to provide a pressure-seal between the housing and the NMR tube16 on the load-bearing side. The NMR tube 16 is 6.150″ long and has adiameter of 0.315″ with a flanged end. Flange 52 is 0.300″ high with adiameter of 0.500″ which allows for a nominal clearance of 0.0015″between the flange and the housing when the NMR tube is placed insidethe housing. The surface of the flange is ground to <0.001″ to provide auniform surface to seal against. Main section 50 of the NMR tube ofdiameter 0.315″ provides a nominal clearance of 0.0015″ with the secondbore section 30. Groove 38 has an O-ring installed in it to form a sealbetween the NMR tube flange 52 and the Be—Cu housing 12. This O-ringseal is critical and must fill the groove to approximately 95% whenassembled. Moreover, preferably, there should be no more than 0.001″ ofspace between third bore section 32 and flange 52 to avoid extrusion ofthe O-ring. The height of the step 60 at the end of cover 14 isnominally 0.200″. These dimensions are provided for illustrativepurposes only and variations will be apparent to those skilled in theart.

The tube is of the same general design as a high pressure sapphire tubedisclosed in patent application Ser. No. 08/967,996, which disclosure isherein incorporated by reference, with a duplicate seal system involvingO-rings embedded in a custom machined BeCu collar and a boron nitridewasher (see FIG. 6). This seal system self-seals under pressure. The NMRcell assembly is pressurized via a small diameter stainless steel tubeleading from the bore of the magnet to the pressure generator, which inthis case will be a supercritical fluid pump. This arrangement is veryeasy to manipulate and control. It should be mentioned that although thepressurization is effectively “gaseous”,in contrast to the liquidpressurization of our high. pressure cell (Urbauer et al., 1996), it isnot as explosive as might be first be imagined. First, the volumes andpressures are small. Second, the liquid alkanes will lose considerableenergy upon vaporization resulting from a sudden pressure drop(Joule-Thompson effect). The combustion risk is al so very low as VTcontrol is done with nitrogen gas, not dry air, so there will benegligible oxygen available in the bore of magnet.

EXAMPLE 5

This embodiment illustrates the use of NMR technology to the reversemicelles of proteins prepared as in Example 1 and also demonstrates thatencapsulation of proteins in reverse micelles does not significantlyalter their native structure. A high resolution NMR, cell capable ofsafe operation at pressures up to 150 bar in an 8 mm triple resonanceprobe was used. This assembly can be used to characterize the NMRproperties of the various proteins under a range of conditions andthereby demonstrate the method of the present invention.

In one illustration of this embodiment, ¹⁵N-HSQC spectra was obtained inan 8 mm probe in a standard tube at 750 MHz for ubiquitin that wasrecombinantly enriched with 13C according to the method of Wand et al.,(1996, Biochemistry, 35:6116-6125), which method is herein incorporatedby reference. introduced into reverse micelles of AOT in pentane. Thespectrum was processed to 1024×1024. The concentration of ubiquitin was0.25 mM. Hydrodynamic diffusion studies confirmed that there was asingle protein molecule per reverse micelle. As shown in FIG. 10,detailed inspection of the spectrum reveals numerous small chemicalshift-changes relative to the equivalent spectrum obtained from anaqueous sample. While not intending to be bound by any particulartheory, it is believed that these chemical shift changes are not due tostructural differences but represent chemical shift changes broughtabout by the large electric fields known to exist at the interface ofsuch reverse micelles. Comparison of the chemical shifts of amide ¹H and¹⁵N in the AOT reverse-micelle and those in water reveals minimaldifferences (FIGS. 11a and 11 b). Including all data, the standarddeviations between amide ¹H and ¹⁵N chemical shifts of ubiquitin in thetwo states are 0.17 and 1.6 ppm respectively. Two localized regionsdisplay perturbations. These include the C-terminal residues R72 and R74and residues 45 to 48 that form a tight turn. The ¹⁵N-resolved NOEspectrum of ubiquitin in AOT/pentane displays the same pattern ofnuclear Overhauser effects among main chain hydrogens as observed whenthe protein is dissolved directly in water (DiStefano et al., 1987,Biochemistry, 31:3645-3652.). These data demonstrates that the method ofencapsulation of proteins as disclosed in the present invention does notintroduce any significant distortion of their native structure.

It should be understood that the examples disclosed herein are forillustrative purposes only and other modifications of the embodiments ofthe present invention that are obvious to those skilled in the art areintended to be within the scope of the present invention.

We claim:
 1. A Pressure cell for use in NMR spectroscopy of moleculescomprising: (a) a sample tube; (b) a housing for holding the sampletube; and (c) a cover for closing the housing, said cover, having atleast one opening for inflow of fluid and at least one opening foroutflow of fluid, wherein the sample tube self-seals under pressureagainst the housing and wherein the cover self-seals under pressureagainst the housing.
 2. The pressure cell of claim 1, wherein the sampletube self-seals under pressure against the housing by an O-ring and awasher.
 3. The pressure cell of claim 1, wherein the cover self-sealsunder pressure against the housing by an O-ring.